DNA double-strand breaks (DSB) can arise by ionizing radiation, alkylation damage, and replication, including improper processing of lagging strand intermediates. DNA breaks can be a powerful source of chromosome instability as well as programmed genetic modification. Cells have elaborate systems for dealing with DSBs, including DNA repair and checkpoint arrest to increase the opportunity for repair. DSBs in chromosomes lead to a checkpoint arrest at the G2/M boundary in yeast, which provides further opportunities for repair. DSBs are repaired through homologous recombination , end-joining , and by single-strand annealing at homologous regions beyond the breaks . Nearly all organisms exhibit these repair processes as well as checkpoint arrests. Defects in these processes are often associated with disease in humans. DNA ends must be processed to allow homologous interactions for recombination and single strand annealing. Endjoining involves only local nuclease degradation that enables interaction at microhomologies of only a few bases, The Ku and RAD50/MRE11/XRS2 (R/M/X) complexes of proteins are required for endjoining. In addition, the R/M/X complex functions in the nuclease processing of ends to provide recombination substrates. The R/M/X complex has been proposed to have a structural role that holds broken chromosomes and sister chromatids together through a Rad50/Mre11 hook-bridge structure. The Ku complex, which associates at the ends of breaks prevents excessive processing of broken ends. The balance of Ku and R/M/X can determine the extent and timing of end-processing of DSBs that in turn determines the timing of G2/M arrest adapation to a DSB. We have investigated the consequences of DSBs in various mutants and the mechanisms of handling DSBs. DSBs IN REAL TIME -- In spite of many genetic and biochemical assays for checkpoint arrest and repair, little is known about the behavior of damaged chromosomes in the arrested cells. Furthermore, the question of the relationship between a DSB in DNA and a cytologically detectable chromosome break, as well as possible genetic controls have never been addressed. We developed a system based in the yeast Saccharomyces cerevisiae that provides for chromosome analysis in real time following the induction of a single DSB by an I-SceI endonuclease under the tight control of a GAL1promoter. We utilized tetR-CFP and LacI-GFP to mark each side of a DSB and Spc29-RFP fusion to identify the spindle poles. These proteins bind multiple repeats of their operater target sequences. This allowed us to investigate the development of a chromosome break following DNA scission and the relation to spindle pole separation and sister chromatid separation in wild type and various Ku and R/M/X mutants. We establish that the transition from DNA double-strand break measured at the molecular level to cytologically detectable chromosome break is prevented by the R/M/X complex. CONSEQUENCES OF VARIOUS KINDS OF DSBS --Double-strand breaks can arise in many ways including, radiation, processing of lesions and even during replication. They can be repaired by homologous recombination (HR) and by nonhomologous endjoing, processes which are common to all eukaryotes and play important roles in various aspects of chromosome metabolism. All cells possess checkpoint systems that enable cells to detect damage and delay cell cycle progression, allowing more time for repair. We have been investigating several aspects of repair relating to defined breaks produced by restriction enzymes and random "dirty" breaks produced by ionizing radiation. For defined DSBs produced by restriction enzymes, the enzymes are placed under the control of a regulatable GAL1 promoter. Growth on galactose leads to the induction of the enzyme in our yeast systems. Previously we showed that in vivo induction of restriction enzyme EcoR I in yeast caused accumulation of breaks that could be repaired not only in wild type yeast, but also in mutants deficient for homologous recombination . It was shown that these EcoR I induced breaks, which have overlapping 5? single strand ends, were being repaired primarily by the nonhomologous end joining pathway. NHEJ deficient yeast had low survival upon induction of EcoR I. We wanted to address the impact of different kinds of breaks, particularly blunt end breaks, since repair and genetic consequences might differ, depending on the nature of the break (i.e., the ends may determine the means). We, therefore, examined the impact of low level induction of the blunt end restriction enzymes PvuII and EcoR5 and compared this to radiation-induced breaks. We show that there are dramatic differences. The system that we used to address breaks and repair, transverse alternating pulse-field electrophoresis (TAFE), allows us to examine changes in full length chromosomes. Therefore, change in specific chromosomes could be addressed as well as the consequences of low doses. This has enabled us to address the impact of the gene RAD9, known to be involved in checkpoint control, in DSB repair.